Reagents and methods for detecting analytes

ABSTRACT

A reagent for detecting an analyte comprises a flavoprotein enzyme, a mediator such as a phenothiazine mediator, at least one surfactant, a polymer and a buffer. The reagent may be used with an electrochemical test sensor that includes a plurality of electrodes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.61/007,126, filed Dec. 10, 2007, the contents of which are incorporatedentirely herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to reagents, methods, anddevices for measurement of analytes. More particularly, the presentinvention relates to reagents, methods, and devices for the measurementof glucose in a blood sample.

BACKGROUND OF THE INVENTION

The quantitative determination of analytes in body fluids is of greatimportance in the diagnoses and maintenance of certain physiologicalphysical conditions. For example, lactate, cholesterol, and bilirubinshould be monitored in certain individuals. In particular, it isimportant that individuals who have diabetes frequently check theglucose level in their body fluids to regulate the glucose intake intheir diets. The results of such tests may be used to determine what, ifany, insulin or other medication should be administered. In one type ofblood-glucose testing system, test sensors are used to test a sample ofblood.

A test sensor contains biosensing or reagent material that reacts with,for example, blood glucose. The testing end of the sensor is adapted tobe placed into the fluid being tested (e.g., blood that has accumulatedon a person's finger after the finger has been pricked). The fluid maybe drawn into a capillary channel that extends in the sensor from thetesting end to the reagent material by capillary action so that asufficient amount of fluid to be tested is drawn into the sensor. Thetests are typically performed using optical or electrochemical testingmethods.

Electrochemical test sensors are based on enzyme-catalyzed chemicalreactions involving the analyte of interest. In the case of glucosemonitoring, the relevant chemical reaction is the oxidation of glucoseto gluconolactone or its corresponding acid. This oxidation is catalyzedby a variety of enzymes; some of which may use coenzymes such asnicotinamide adenine dinucleotide (phosphate) (NAD(P)), while others mayuse coenzymes such as flavin adenine dinucleotide (FAD) orpyrroloquinolinequinone (PQQ).

In test sensor applications, the redox equivalents generated in thecourse of the oxidation of glucose are transported to the surface of anelectrode, whereby an electrical signal is generated. The magnitude ofthe electrical signal is then correlated with glucose concentration. Thetransfer of redox equivalents from the site of chemical reaction in theenzyme to the surface of the electrode is accomplished using electrontransfer mediators.

Electron transfer mediators previously used with FAD-glucosedehydrogenase (FAD-GDH) include potassium ferricyanide,phenazine-methosulfate (PMS), methoxy phenazine-methosulfate, phenazinemethyl sulfate, and dichloroindophenol (DCIP). These compounds, however,have proven to be highly susceptible to the environmental conditionsincluding temperature and moisture, which result in test sensor reagentsof low stability. For example, during storage, reduced mediator may beproduced from interactions between the oxidized mediator and the enzymesystem. The larger the amount of mediator or enzyme, the larger theamount of reduced mediator that is produced. The background current,which increases over time, will generally increase toward the end of theshelf-life of the sensor strips because of the high concentration ofreduced mediator. The increased background current may decrease theprecision and accuracy of the measurements of the test sensor and, thus,provide a limited shelf-life for the test sensors.

Another disadvantage associated with existing test sensors is therelatively slow fill rate. Achieving a fast sensor fill rate isdesirable so that the re-hydration of the reagent may be faster and moreuniform. Thus, faster fill rates generally result in more precise,stable test sensors having less variation.

Therefore, it would be desirable to have a reagent that addresses one ormore of these disadvantages.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a reagent fordetecting an analyte comprises a flavoprotein enzyme, a mediatorselected from the group

or a combination thereof. R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are thesame or different and are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy,aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, andcombinations thereof. The reagent further comprises at least onesurfactant, a polymer and a buffer. At least one of the surfactant andthe buffer includes an inorganic salt in which the ratio of the totalinorganic salt to mediator is less than about 3:1.

According to another embodiment of the present invention, a reagent fordetecting an analyte in a fluid sample includes FAD-glucosedehydrogenase having an activity of from about 0.1 Units/μL to about 10Units/μL. The reagent further comprises a3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator having aconcentration of from about 5 mM to about 120 mM. The reagent furthercomprises a surfactant having a concentration of from about 0.05 wt. %to about 0.5 wt. % of the reagent. The reagent further comprises ahydroxyethyl cellulose polymer having a concentration of from about 0.1wt. % to about 4 wt. % of the reagent and a buffer. At least one of thesurfactant and the buffer includes an inorganic salt in which the ratioof the total inorganic salt to mediator is less than about 3:1.

According to another embodiment of the present invention, anelectrochemical test sensor comprises a working electrode having asurface. The test sensor further comprises a counter electrode having asurface. The test sensor further comprises a reagent coating at least aportion of the surface of the working electrode and at least a portionof the surface of the counter electrode. The reagent comprises aflavoprotein, a phenothiazine or a phenoxazine mediator, a buffer, andat least one surfactant and a polymer. At least one of the surfactantand the buffer includes an inorganic salt in which the ratio of thetotal inorganic salt to mediator is less than about 3:1.

According to one process of the present invention, a method of detectingan analyte in a fluid sample, the analyte undergoing a chemicalreaction, comprises the act of providing an electrode surface. Themethod further comprises the act of facilitating flow of the fluidsample to the electrode surface using a surfactant. The method furthercomprises the act of catalyzing the chemical reaction with aflavoprotein enzyme. The method further comprising the act of generatinga redox equivalent by the chemical reaction. The method furthercomprises the act of transferring the redox equivalent to the electrodesurface using a phenothiazine or a phenoxazine mediator. The maximumkinetic performance is less than about 3 seconds.

According to another method, an analyte is detected in a fluid sampleand includes providing an electrode surface. A reagent is provided thatincludes a flavoprotein enzyme and a mediator is selected from the group

or a combination thereof, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹are the same or different, and are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy,aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, andcombinations thereof; at least one surfactant; and a buffer; the reagentcontacting the electrode surface. The fluid sample contacts the reagent.The concentration of the analyte is determined. The maximum kineticperformance is less than about 3 seconds.

The above summary of the present invention is not intended to representeach embodiment, or every aspect, of the present invention. Additionalfeatures and benefits of the present invention are apparent from thedetailed description and figures set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a test sensor according to one embodiment.

FIG. 1 b is a side view of the test sensor of FIG. 1 a.

FIG. 2 is a line graph plotting current measurements against glucoseconcentrations.

FIG. 3 is a bar graph comparing fill times of sensors includingheptanoyl-N-methylglucamide (MEGA 8) surfactant with sensors notincluding MEGA 8 surfactant.

FIG. 4 is a bar graph comparing background current of sensors includingMEGA 8 surfactant with sensors not including MEGA 8 surfactant.

FIG. 5 is a bar graph of fill times of test sensors versus differentformulations with and without surfactants.

FIG. 6 is a plot of measured current values versus time in a formulationincluding a surfactant.

FIG. 7 is a bar graph of peak times using 50 mg/dL of glucose, mediatorswith different sulfate concentrations and different phosphate bufferconcentrations.

FIG. 8 is a bar graph of peak times using 100 mg/dL of glucose,mediators with different sulfate concentrations and different phosphatebuffer concentrations.

FIG. 9 is a bar graph of peak times using 400 mg/dL of glucose,mediators with different sulfate concentrations and different phosphatebuffer concentrations.

FIGS. 10 a, 10 b are plots of measured current values versus time informulations having different inorganic salt concentrations.

FIG. 11 is a graph with % CV for low salt reagent solutions and highsalt reagent formulations.

While the invention is susceptible to various modifications andalternative forms, specific embodiments are shown by way of example inthe drawings and are described in detail herein. It should beunderstood, however, that the invention is not intended to be limited tothe particular forms disclosed. Rather, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

DESCRIPTION OF ILLUSTRATED EMBODIMENTS

The present invention is directed to reagents, methods, and devices formeasurement of analytes. More specifically, the present invention isdirected to a test sensor reagent for detecting an analyte including (1)a flavoprotein enzyme, (2) a phenothiazine or phenoxazine mediator, (3)a buffer, (4) a surfactant or a combination of surfactants, and/or (5) acellulose-based polymer.

The reagents described herein may be used to assist in determining ananalyte concentration in a fluid sample. The nature of the analytemonitored in accord with the present invention is unrestricted, providedthe analyte undergoes a chemical reaction that is catalyzed by aflavoprotein enzyme. Some examples of the types of analytes that may becollected and analyzed include glucose, lipid profiles (e.g.,cholesterol, triglycerides, LDL, and HDL), microalbumin, hemoglobin,A_(1C), fructose, lactate, or bilirubin. It is contemplated that otheranalyte concentrations may be determined. The analytes may be in, forexample, a whole blood sample, a blood serum sample, a blood plasmasample, other body fluids such as ISF (interstitial fluid), urine, andnon-body fluids.

The test sensors described herein are electrochemical test sensors.Meters used with the electrochemical test sensors may have opticalaspects so as to detect the calibration information and electrochemicalaspects to determine information related to the analyte (e.g., theanalyte concentration) of the fluid sample. One non-limiting example ofan electrochemical test sensor is shown in FIG. 1 a. FIG. 1 a depicts atest sensor 10 including a base 11, a capillary channel, and a pluralityof electrodes 16 and 18. A region 12 shows an area that defines thecapillary channel (e.g., after a lid is placed over the base 11). Theplurality of electrodes includes a counter electrode 16 and a workingelectrode 18. The electrochemical test sensor may also contain at leastthree electrodes, such as a working electrode, a counter electrode, atrigger electrode, or another electrode to detect interferencesubstances (e.g., hematocrit, ascorbate, uric acid) in the fluid sample.The working electrode employed in electrochemical sensors according tothe embodiments of the present invention may vary, with suitableelectrodes including, but not limited to, carbon, platinum, palladium,gold, combinations thereof, and the like.

The electrodes 16, 18 are coupled to a plurality of conductive leads 15a,b, which, in the illustrated embodiment, terminates with larger areasdesignated as test-sensor contacts 14 a,b. The capillary channel isgenerally located in a fluid-receiving area 19. Examples ofelectrochemical test sensors, including their operation, may be foundin, for example, U.S. Pat. No. 6,531,040 assigned to Bayer Corporation.It is contemplated that other electrochemical test sensors may beemployed with the embodiments of the present invention.

The fluid-receiving area 19 includes at least one reagent for convertingthe analyte of interest (e.g., glucose) in the fluid sample (e.g.,blood) into a chemical species that is electrochemically measurable, interms of the electrical current it produces, by the components of theelectrode pattern. The reagent typically includes an analyte-specificenzyme that reacts with the analyte and with an electron acceptor toproduce an electrochemically measurable species that may be detected bythe electrodes. The reagent may include mediators or other substancesthat assist in transferring electrons between the analyte and theconductor, binders that hold the enzyme and mediator together, otherinert ingredients, or combinations thereof.

A fluid sample (e.g., blood) may be applied to the fluid-receiving area19. The fluid sample reacts with the at least one reagent. Afterreacting with the reagent and in conjunction with the plurality ofelectrodes, the fluid sample produces electrical signals that assist indetermining the analyte concentration. The conductive leads 15 a,b carrythe electrical signal back toward a second opposing end 42 of the testsensor 10 where the test-sensor contacts 14 a,b transfer the electricalsignals into the meter.

Referring to FIG. 1 b, a side view of the test sensor 10 of FIG. 1 a isshown. As shown in FIG. 1 b, the test sensor 10 of FIG. 1 b furtherincludes a lid 20 and a spacer 22. The base 11, the lid 20, and thespacer 22 may be made from a variety of materials such as polymericmaterials. Non-limiting examples of polymeric materials that may be usedto form the base 11, the lid 20, and the spacer 22 includepolycarbonate, polyethylene terephthalate (PET), polyethylenenaphthalate (PEN), polyimide, and combinations thereof. It iscontemplated that other materials may be used in forming the base 11,lid 20, and/or spacer 22.

To form the test sensor 10 of FIGS. 1 a, 1 b, the base 11, the spacer22, and the lid 20 are attached by, for example, an adhesive or heatsealing. When the base 11, the lid 20, and the spacer 22 are attached,the fluid-receiving area 19 is formed. The fluid-receiving area 19provides a flow path for introducing the fluid sample into the testsensor 10. The fluid-receiving area 19 is formed at a first end ortesting end 40 of the test sensor 10. Test sensors of the embodiments ofthe present invention may be formed with a base and a lid in the absenceof a spacer, where the fluid-receiving area is formed directly in thebase and/or the lid.

Flavoproteins in accord with the present invention include any enzymeshaving flavin cofactors. Some non-limiting examples of flavoproteinsinclude FAD-glucose oxidase (Enzyme Classification No. 1.1.3.4),Flavin-hexose oxidase (EC No. 1.1.3.5) and FAD-glucose dehydrogenase (ECNo. 1.1.99.10). Additional oxidase enzymes for use in accord with thepresent invention include, but are not limited to, lactate oxidase,cholesterol oxidase, alcohol oxidase (e.g., methanol oxidase),d-aminoacid oxidase, choline oxidase, and FAD derivatives thereof Adesirable flavoprotein for use in accord with the present invention isFAD-glucose dehydrogenase (FAD-GDH).

Mediators in accord with the present invention include phenothiazineshaving the formula

and phenoxazines having the formula

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or differentand are independently selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo,haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl,aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino,aminoalkyl, sulfo, dihydroxyboron, and combinations thereof. It iscontemplated that isomers of the same may also be formed.

One desirable example of a phenothiazine that has been prepared andfound to have suitable properties as an NADH mediator is a water-solublesodium or ammonium salt of 3-(2′,5′ disulfophenylimino)-3H-phenothiazinehaving the formula

3-(2′,5′-disulfophenylimino)-3H-phenothiazine is associated with aparticularly low background current, which results in improvedsignal-to-noise ratios. Another desirable example is3-(3′,5′-dicarboxy-phenylimino)-3H-phenothiazine mediator that has beenprepared and found to have suitable properties as an NADH mediator. Thebackground current of these phenothiazines was found to be significantlyless than previously-used mediators.

Other phenothiazines and phenoxazines that have been found to havesuitable properties as NADH mediators are3-(4′-chloro-phenylimino)-3H-phenothiazine;3-(4′-diethylamino-phenylimino)-3H-phenothiazine;3-(4′-ethyl-phenylimino)-3H-phenothiazine;3-(4′-trifluoromethyl-phenylimino)-3H-phenothiazine;3-(4′-methoxycarbonyl-phenylimino)-3H-phenothiazine;3-(4′-nitro-phenylimino-3H-phenothiazine;3-(4′-methoxy-phenylimino)-3H-phenothiazine;7-acetyl-3-(4′-methoxycarbonylphenylimino)-3H-phenothiazine;7-trifluoromethyl-3-(4′-methoxycarbonyl-phenylimino)-3H-phenothiazine;3-(4′-ω-carboxy-n-butyl-phenylimino)-3H-phenothiazine;3-(4′-aminomethyl-phenylimino)-3H-phenothiazine;3-(4′-(2″-(5″-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine;3-(4′-p-aminoethyl-phenylimino)-3H-phenothiazine;6-(4′-ethylphenyl)amino-3-(4′-ethylphenylimino)-3H-phenothiazine;6-(4′-[2-(2-ethanoloxy)ethoxy]-ethoxyphenyl)amino-3-(4′-[2-(2-ethanoloxy)ethoxy]ethoxyphenylimino)-3H-phenothiazine;3-(4′-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino)-3H-phenothiazine;3-(4′-phenylimino)-3H-phenothiazineboronic acid,3-(3′,5′-dicarboxy-phenylimino)-3H-phenothiazine;3-(4′-carboxyphenylimino)-3H-phenothiazine;3-(3′,5-dicarboxy-phenylimino)-3H-phenoxazine;3-(2′,5′-phenylimino)-3H-phenothiazinedisulfonic acid; and3-(3′-phenylimino)-3H-phenothiazinesulfonic acid.

In one embodiment, a 3-(2′,5′-disulfophenylimino)-3H-phenothiazinemediator was prepared by dissolving phenothiazine (1.53 mole, 1.1equivalent, 306 g) with stirring into 6.0 L of tetrahydrofuran (THF) andthen cooled to 0° C. Aniline 2,5-disulfonic acid (1.38 mole, 350 g) wasdissolved in 7.0 L of water and 1 M sodium hydroxide (NaOH) (128 ml) wasadded during stirring. The aniline 2,5-disulfonic acid solution wasadded slowly, over the course of about 2 hrs, to the phenothiazinesolution, to give a white, cloudy suspension. The phenothiazine/anilinesuspension was at a temperature of about 0° C. to about 4° C. Sodiumpersulfate (5.52 mole, 4 equivalent, 1314 g) was dissolved in 4.0 L ofwater to form a sodium persulfate solution.

The sodium persulfate solution was added dropwise over 3 hours to thephenothiazine/aniline suspension at a temperature between about 0° C. toabout 3° C. and resulted in a very dark solution. The very dark solutionremained cold using an ice bath and was stirred overnight. The contentswere then transferred to a Buchi rotary evaporator and thetetrahydrofuran was removed over the course of about 2 hours at atemperature less than 35° C. After the evaporation act, the remainingsolution was transferred to a 25 L separator and backwashed with ethylacetate. The remaining solution was backwashed 3 times using 2 L ofethyl acetate each time. The reaction fluids were cooled while stirringto −3° C. in an acetone/CO₂ bath. The precipitated solid was filteredthrough two cloths on two 24 cm Buchner funnels on the same day. Theprecipitated solid was left overnight in the funnels to dry and thentransferred to a flask containing 2 L of acetonitrile and stirred forabout 1 hour at room temperature. To remove the residual water, thesample was then filtered and washed with more acetonitrile. The mediatorwas dried to a constant weight in a vacuum oven at 35° C.

Because of the low background current achieved using reagents having3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediators, the samereagent formulation may be applied to both the working electrode and thecounter electrode of an electrochemical test sensor. Applying the samereagent to both the working electrode and the counter electrodesimplifies the manufacturing process and thereby decrease the costsassociated therewith. Additionally, the low background current assistsin obtaining accurate glucose readings, especially with samples havinglow glucose concentrations, which is particularly important in analyzingneonatal blood glucose assays.

The reagents of the embodiments of the present invention further includea surfactant or a combination of surfactants, and/or a cellulose-basedpolymer. The surfactant or combination of surfactants facilitates thesensor blood fill rate and re-hydration of a dry reagent. The fasterblood fill rate and reagent re-hydration rate are desirable forachieving a quicker assay (e.g., less than 5-second assay) across anabout 20% to an about 70% hematocrit range.

The surfactant is desirably selected from biocompatible ones includingsaccharide-based surfactants or phosphorylcholine-based surfactants. Onenon-limiting example of a saccharide-based surfactant isheptanoyl-N-methylglucamide (MEGA 8 from Sigma-Aldrich of St. Louis,Mo.). Surfactants such as MEGA 8 assist in increasing the thermalstability of test sensors. Additionally, surfactants such as MEGA 8assist in fast fill rates, even for blood samples having high hematocritlevels. Using surfactants such as MEGA 8 with other inert ingredients,(e.g., hydroxyethyl cellulose polymer and/or a neutral pH buffer) in areagent formulation provides sensors with great stability, even atelevated temperatures. Non-limiting examples of phosphorylcholine-basedsurfactants include the Lipidure series (NOF Corporation, Japan).

Surfactants may also be selected from conventional neutral surfactantssuch as ethoxylated oleyl alcohol (Rhodasurf ON870 from Rhodia Inc. inCranbury, N.J.). Surfactants may also be selected from anionicsurfactants such as sodium methyl cocoyl taurate (Geropon TC-42 fromRhodia Inc.) and alkyl phenol ethoxylate phosphate (Phospholan CS131from Akzo-Nobel Surface Chemistry LLC in Chicago, Ill.). It iscontemplated that other surfactants may be used in forming the reagent.

Alternatively or additionally, the reagents of the embodiments of thepresent invention include a polymer. The reagents may include acellulose-based polymer such as hydroxyethyl cellulose polymer. In someembodiments, the cellulose-based polymer is a low to medium molecularweight cellulose-based polymer. The polymer, such as a cellulose-basedpolymer, assists in providing the reagent with increased stability andadequate viscosity so that the reagent, when dried, stays in itsoriginal position on the sensor substrate. It is contemplated that otherpolymers may be used such as, for example, polyvinyl pyrrolidine (PVP).

The reagent may also include a buffer (e.g., a phosphate buffer) and/orother inert components. Non-limiting examples of suitable buffersolutions include but are not limited to Good's buffers (e.g., HEPES(i.e., N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), MOPS (i.e.,3-(N-morpholino)propanesulfonic acid), TES (i.e.,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid)), Mcllvaine'sbuffers, combinations thereof, or the like.

To provide a desirable assay precision, thermal stability and maximumkinetic performance, the ratio of the inorganic salts to mediator shouldbe less than about 3:1. The source of inorganic salts could be from thebuffer and/or the mediator. It is even more desirable for the ratio ofthe inorganic salts to mediator to be less than about 2:1 or even lessthan about 1.5:1.

According to one embodiment of the present invention, a reagent includesFAD-GDH, a low background phenothiazine mediator, a surfactant orcombination of surfactants, a cellulose-based polymer, and a buffer toachieve improved sensor performance and stability. The reagent may beused to determine the glucose concentration in biological specimen suchas blood, plasma, serum, or urine. In one embodiment, the phenothiazinemediator is 3-(2′,5′-disulfophenylimino)-3H-phenothiazine. In anotherembodiment, the surfactant is MEGA 8 and the polymer is hydroxyethylcellulose. In one embodiment, a reagent includes FAD-GDH having anactivity ranging from about 0.1 Units/μL to about 10 Units/μL, about 5mM to about 120 mM of 3-(2′,5′-disulfophenylimino)-3H-phenothiazinemediator, about 0.05 wt. % to about 0.5 wt. % of MEGA 8 surfactant,about 0.1 wt. % to about 4 wt. % of hydroxyethyl cellulose, and about 25mM to about 200 mM of buffer having a pH of about 4 to about 8. Inanother embodiment, a reagent includes FAD-GDH having an activityranging from about 0.5 Units/μL to about 2.5 Units/μL, about 30 mM toabout 60 mM of 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator,about 0.1 wt. % to about 0.4 wt. % of MEGA 8 surfactant, 0.01 to 0.1% ofGeropon TC-42, about 0.2 wt. % to about 0.5 wt. % of hydroxyethylcellulose, and about 50 mM to about 150 mM of buffer having a pH ofabout 6 to about 7.

Example 1

As shown in FIG. 2, the reactivity of the chemistry for four sensor lotswas analyzed by generating a glucose dose-response curve for sensorsincluding FAD-GDH enzyme having an activity of about 1.75 Units/μL,about 40 mM 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator,about 0.2 wt. % MEGA 8 surfactant, about 0.25 wt. % hydroxyethylcellulose, and about 100 mM phosphate buffer having a pH of about 6.5.The sensors were tested with whole blood samples at about 40% hematocritlevel. The blood glucose concentrations of the blood samples were about0 mg/dL, 38 mg/dL, 67 mg/dL, 112 mg/dL, 222 mg/dL, 339 mg/dL, and 622mg/dL. For each blood sample, ten replicates were collected for eachsensor lot. As shown in FIG. 2, the mean current for each sample wasplotted against the sample glucose concentration (mg/dL) measured by aYellow Springs Glucose Analyzer (YSI, Inc., Yellow Springs, Ohio) foreach sensor lot. The slope of the dose response lines was about 20nA/mg/dL, which indicates relatively high sensitivity of the testsensors. The y-intercepts were relatively close to 0 nA, which indicateslow background noise levels. These results indicate that accuratereadings may be achieved using the test sensors including the reagentdescribed herein.

The coefficient of variation was determined for each of the tenreplicates of the sensor lots used to generate the graph of FIG. 2.Table 1 below shows the average coefficient of variation percent (% CV)from the four sensor lots.

TABLE 1 Glucose Concentration 38 67 112 222 339 622 mg/dL mg/dL mg/dLmg/dL mg/dL mg/dL % CV 3.1 2.1 3.4 2.5 2.1 1.2

Because of the low background noise of the sensors including the reagentof the embodiments of the present invention, the average assay % CV wasless than 3.5%, even for samples having low glucose concentrations.Thus, the % CV values were well under 5%, which is often considered tobe the standard acceptable limit. This low % CV indicates high precisionof the test sensors. Additionally, the low % CV is associated with lowvariance among test sensors, which is desirable for obtaining consistenttest results.

Example 2

FIG. 3 shows a graph illustrating the affect of MEGA 8 surfactant on thesensor fill rate using 60% hematocrit whole blood. The test sensors usedin FIG. 3 included FAD-GDH having an activity of about 1 Unit/μL (about192 Units/mg), about 4 wt. % (about 120 mM) of potassium ferricyanidemediator, about 1.6 wt. % of the reagent of 4 wt. % hydroxyethylcellulose, and about 35 mM of citrate buffer at a pH of about 5.0.Potassium ferricyanide mediator was used to test whether MEGA 8surfactant without 3-(2′,5′-disulfophenylimino)-3H-phenothiazinemediator had desirable affects on a test sensor. The fill rates of agroup of thirty test sensors including about 0.2 wt. % MEGA 8 surfactantwere compared against a control group of thirty test sensors notincluding MEGA 8 surfactant. Initial fill rates of ten sensors from eachof the two groups of were measured. Ten sensors from each group werethen exposed to a temperature of about −20° C. for about two weeks.Finally, ten sensors from each group were exposed to a temperature ofabout 50° C. for about two weeks. The average fill times of each groupand subgroup of test sensors were calculated and are shown in FIG. 3.The blood fills the reaction chamber of the test sensor in less than 0.3sec. with the reagent having surfactant and a 60% hematocrit whole bloodsample. As shown in FIG. 3, the fill rate of the sensors including MEGA8 surfactant was at least twice as fast and up to about four timesfaster than those sensors not including the MEGA 8 surfactant.

Example 3

The background currents of heat-stressed sensors including MEGA 8surfactant were compared to the background currents of heat-stressedsensors not including MEGA 8 surfactant. The test sensors used in FIG. 4included FAD-GDH having an activity of about 1 Unit/μL, about 50 mM of3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator, about 0.75 wt. %of the reagent of hydroxyethyl cellulose, and about 50 mM of buffer at apH of about 7. A first group of forty test sensors did not include MEGA8 surfactant. A second group of forty test sensors included about 0.2wt. % MEGA 8 surfactant. Each of the first and second groups includedtwo subgroups: a first subgroup including twenty test sensors havingFAD-GDH from Amano Enzyme Inc. (Nagoya, Japan) and a second subgroupincluding twenty test sensors having FAD-GDH from Toyobo Co. (Osaka,Japan) Ten of the test sensors from each of the subgroups were stored atabout 50° C. for about two weeks. The remaining test sensors were storedat about −20° C. for about two weeks. The background current of thesensors was then tested using 40% hematocrit whole blood samples havinga glucose concentration of about 0 mg/dL. Ten replicates per sample werecollected. FIG. 4 shows a graph illustrating the mean sensor backgroundcurrent from the ten replicates. As shown in FIG. 4, the test sensorsincluding MEGA 8 surfactant had a significantly lower sensor backgroundcurrent change as compared with the test sensors not including MEGA 8surfactant, indicating that the reagent is more stable when MEGA 8surfactant is added to the reagent.

Example 4

The thermal stability of test sensors according to the embodiments ofthe present invention were also tested. The test sensors used in thisexample included FAD-GDH having an activity of about 2 Units/μL, about40 mM of 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator, about0.2 wt. % of MEGA 8 surfactant, about 0.25 wt. % of the reagent ofhydroxyethyl cellulose, and about 100 mM of buffer at a pH of about 6.5.A first group of test sensors was stored at about 50° C. for about twoweeks. A second group of test sensors was stored at about −20° C. forabout two weeks. The performance of the sensors in each group was thenevaluated with 40% hematocrit whole blood samples having glucoseconcentrations of about 50 mg/dL, about 100 mg/dL, and about 400 mg/dL.Ten replicates per sample were collected. The mean difference in glucoseconcentration between the test sensors stored at 50° C. and those storedat −20° C. was calculated and compared to several different types ofself testing blood glucose monitoring systems. The glucose assay bias ofthe test sensors according to the embodiments of the present inventionwas negligible. Thus, there was no appreciable change in the glucoseassay results even after storing the sensors at relatively extremetemperatures for two weeks. In contrast, the glucose assay bias of thecomparative commercially available test sensors was generally from about5% to about 12%. Thus, the thermal stability of the test sensors of theembodiments of the present invention was significantly better than thatof existing test sensors.

Example 5

Tests were performed using test sensors to determine fill speed withhigh hematocrit blood samples. Specifically, as shown in FIG. 5, aformulation using no surfactant (Formula 1) and surfactants (Formulas2-7) were tested. The formulations are listed in Table 2 below.

TABLE 2 Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6Formula 7 Mediator (mM) 60 60 60 60 60 50 90 Buffer (mM) 75 75 75 75 75100 112 FAD-GDH (U/uL) 3.00 3.00 3.00 3.00 3.00 1.25 3.75 Polymer(HEC)(%) 0.38 0.38 0.38 0.38 0.38 0.63 0.60 MEGA 8 (wt. %) 0.10 0.050.225 Rhodasurf (wt. %) 0.10 0.05 Zwittergent 312 (wt. %) 0.30Phospholan CS131 (wt. %) 0.10 Mediator =3-(2′,5′-disulfophenylimino)-3H-phenothiazine Buffer = Phosphate exceptFormula 6 used TES TES =(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) HEC =hydroxyethyl celluloseSpecifically, Formulas 2 and 3 included the surfactant MEGA 8, whileFormulas 4 and 5 included the surfactant Rhodasurf. Formula 6 includedthe surfactant Zwittergent and Formula 7 included surfactants MEGA 8 andPhospholan CS 131. Formulas 1-7 were stressed under two differentconditions. Specifically, Formulas 1-7 were stressed for 2 weeks at atemperature of −20° C. and also stressed for 2 weeks at a temperature of50° C.

After being stressed at these conditions, Formulas 1-7 were depositedonto electrodes on the test sensor. The sensors were tested in avertical (90°) position with whole blood at 60-70% hematocrit. Thesensors were videotaped during filling and the time was measured. Thetimes required for the high hematocrit blood to fill the entire sensorreaction chamber for Formulas 1-7 are shown in FIG. 5. The sensor-filltime for Formula 1 (without surfactant) was about 0.6 and 0.7 sec. forthe sensors stored at −20° C. and 50° C./2 wks., respectively. On thehand, Formulas 2-7 had sensor-fill times for both −20° C. and 50° C./2wks. were about 0.5 sec. or less. Most of the Formulas 2-7 hadsensor-fill times for both −20° C. and 50° C./2 wks. that were about 0.4seconds or less with several being less than about 0.3 sec. Thus,Formulas 2-7 with surfactants had much improved fill times over Formula1 without a surfactant.

Example 6

A reagent was tested to determine its maximum kinetic performance. Thereagent included 40 mM of 3-(2′,5′-disulfophenylimino)-3H-phenothiazine,50 mM of phosphate buffer, 2.00 U/ul of FAD-GDH, 0.25 wt. % ofhydroxyethyl cellulose (HEC) and 0.20 wt. % of the surfactant MEGA 8.

FIG. 6 shows the output signals from test sensors having blood sampleswith a glucose concentration of 400 mg/dL and 70% hematocrit. The signalinput to the sensor strip by the measurement device was a gatedamperometric pulse sequence that included eight pulsed excitationsseparated by seven relaxations, such as described in U.S. PatentPublication No. 2008/0173552. The excitations were less than a 1 secondin duration. Three output current values were recorded during eachexcitation.

To correlate the output current values from the input signal with theanalyte concentration of the sample, the initial current value from theexcitation is preferably greater than those that follow in the decay.The output signals from the sensor strip of FIG. 6 showed an initialhigh current value that decays at about two seconds after the bloodsample was introduced to the strip. Thus, the first output currentshaving a high initial current value followed by decaying current valueswere observed in output current 110.

To correlate the output current values from the input signal to theanalyte concentration of the sample, different sample analyteconcentrations also preferably show a substantially constant differencebetween output signal current values. Preferably, the output currentvalue or values correlated with the analyte concentration of the samplealso are taken from a decay including current data reflecting themaximum kinetic performance of the sensor strip. The kinetics of theredox reaction underlying the output currents is affected by multiplefactors. These factors may include the rate at which the reagentcomposition rehydrates, the rate at which the enzyme system reacts withthe analyte, the rate at which the enzyme system transfers electrons tothe mediator, and the rate at which the mediator transfers electrons tothe electrode. Of these and other kinetic factors affecting the outputcurrents, the rate at which the reagent composition rehydrates isbelieved to have the greatest influence on the output currents.

The maximum kinetic performance of the sensor strip may be reachedduring an excitation of a gated amperometric pulse sequence when theinitial current value of an excitation having decaying current values isgreatest for the multiple excitations. This may also be referred to assensor-peak time. Preferably, the maximum kinetic performance of asensor strip is reached when the last in time current value obtained foran excitation having decaying current values is the greatest last intime current value obtained for the multiple excitations. Morepreferably, the maximum kinetic performance of a sensor strip is reachedwhen the initial current value of an excitation having decaying currentvalues is greatest for the multiple excitations and the last in timecurrent value obtained for the same excitation is the greatest last intime current value obtained for the multiple excitations.

The maximum kinetic performance of the sensor strip is desirably lessthan about 3 seconds and even more desirably less than about 2 seconds.

The gated amperometric pulse sequence used to determine the maximumkinetic performance of a test sensor included at least seven dutycycles, where the excitations are about 0.4 sec. in duration and therelaxations are 1 sec. in duration, include zero current flow throughthe sample, and are provided by an open circuit. At least three outputcurrent values are measured during each excitation. The potential inputto the sensor strip is held substantially constant, at 250 mV and thesample temperature is at 23° C. Before the duty cycles, a pulse of 400mV was applied for 0.9 seconds.

The sensor strip with 400 mg/dL of glucose in FIG. 6 reached maximumkinetic performance during the excitation decay that included outputcurrents 120 and 125, between 3 and 4 seconds from the introduction ofthe sample to the sensor strip. This was established as both thegreatest initial and the greatest last in time current values obtainedfrom an excitation having decaying current values were present in thecycle that included output currents 120 and 125. Compare initial outputcurrent 120 with output currents 110, 130, 140, 150, 160 and 170 andalso compare last in time output current 125 with output currents 115,135, 145, 155, 165 and 175. Thus, the sensor reaches its maximum kineticperformance in between 3 and 4 seconds even for a 70% hematocrit bloodsample

Example 7

FIGS. 7-9 show that the reagent formulation can also affect maximumkinetic performance for blood with different hematocrit levels.Referring initially to FIG. 7, at a glucose concentration of 50 mg/dL,the maximum kinetic performance using sensor-peak time increased as thehematocrit level increased. The mediator that included a smaller sulfatepercentage (5% sulfate) generally had much faster peak times atcomparable hematocrit levels and buffer concentrations. See, forexample, at hematocrit level 60% using 50 mM phosphate buffer (compare3.5 sec. with mediator using 5% sulfate and 7.5 sec. with mediator using20% sulfate). The reagent formulation, such as buffer strength andresidual sulfate content of the mediator, greatly impacts sensorreaction peak time, especially for samples with high hematocrit (>40%).Thus, high inorganic salt (from buffer or from the mediator in terms ofsulfate concentration) in sensor formulation increases sensor-peak time(i.e., slows down sensor reaction). FIG. 8 shows similar results using100 mg/dL of glucose concentration. Using the high glucose concentrationof 400 mg/dL, FIG. 9 shows maximum kinetic performance using sensor-peaktime generally below about 3 or 3.5 seconds with a number of sensor-peaktimes of 2 seconds. The mediator with 5% sulfate and 100 mM of phosphatebuffer at high hematocrit levels had a maximum kinetic performance usingsensor-peak time of about 4.5 or 5 seconds.

Thus, as shown in the 50 mg/dL and 100 mg/dL glucose concentrations, toachieve a fast reagent re-hydration and glucose reaction for sampleswith high hematocrit, the salt content in reagent formulations has to belowered.

Example 8

Two formulations were tested for maximum kinetic performance after thetest sensors had been stored under stressed conditions (−20° C. and 50°C./4 wks.). The formulation in FIG. 10 a included 50 mM phosphate bufferand a 3-(2′,5′-disulfophenylimino)-3H-phenothiazine having 5 wt. %sulfate. The formulation in FIG. 10 b included 100 mM phosphate bufferand a 3-(2′,5′-disulfophenylimino)-3H-phenothiazine having 20 wt. %sulfate. The gated amperometric pulse sequence used in this Example wassimilar to that described in Example 6 above.

As shown in FIG. 10 a, maximum kinetic performance was about 2 secondsfor the samples that were stressed at −20° C. for 4 wks. The maximumkinetic performance was about 3.5 seconds for the samples that werestressed at −50° C. for 4 wks. Referring to FIG. 10 b, the maximumkinetic performance was about 3.5 seconds for the samples that werestressed at −20° C. for 4 wks. and about 5 seconds for the samples thatwere stressed at −50° C. for 4 wks. Thus, formulations having a lowerinorganic salt amount (e.g., FIG. 10 a) had an improved maximum kineticperformance as compared to formulations with a higher inorganic saltamount (e.g., FIG. 10 b).

In addition, lots of the formulation used in FIG. 10 a with 2 secondsensor-peak times were tested for assay bias or % bias. The % bias forglucose concentrations not greater than 100 mg/dL were less than about+/−2%. The % bias for a glucose concentration of 400 mg/dL was about+/−4%. The assay biases for lots of formulation used in FIG. 10 b having3-4 second sensor-peak times were also tested. The % bias for glucoseconcentrations not greater than 100 mg/dL were less than about +/−3%.The % bias for a glucose concentration of 400 mg/dL was about +/−10%.Thus, the assay biases for the lots having 3-4 second sensor-peak timeswere greater than the lots having 2 second sensor-peak times.

Example 9

Referring to FIG. 11, the coefficient of variation % (% CV) using 40%hematocrit whole blood samples at different glucose concentrations areshown. The glucose concentrations range from 36 mg/dL to 627 mg/dL.Reagent solutions having a low salt content lot were compared to reagentsolutions having a high salt content lot. The low salt lot included 50mM phosphate buffer at pH 6.5, 2 U/ul FAD-GDH, 40 mM3-(2′,5′-disulfophenylimino)-3H-phenothiazine having 5 wt. % sulfate,0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8 surfactant. The highsalt lot included 100 mM phosphate buffer at pH 6.5, 2 U/ul FAD-GDH, 40mM 3-(2′,5′-disulfophenylimino)-3H-phenothiazine having 20 wt. %sulfate, 0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8 surfactant.

The % CV was calculated by taking the mean of the maximum kineticperformance using sensor-peak times and dividing by the standarddeviation of those sensor-peak times. This resulting value wasmultiplied by 100, resulting in the % CV. A total of 40 samples weretested for both the low salt reagent solutions and the high salt reagentsolutions.

The low salt reagent solution reached maximum kinetic performance usingsensor-peak times in less than 3 seconds, resulting in a better % CV for40% hematocrit whole blood samples as compared to the higher saltreagent solution. The low salt reagent solutions had a much better % CVat the lower glucose concentration samples.

While the examples provided herein relate to in vitro applications ofthe test sensor reagents in accordance with the present invention, it iscontemplated that these reagents may also be adapted for in vivo analytemonitoring by chemically immobilizing the mediators (e.g., by chemicalreaction at one or more of the substituent groups on the aromaticrings), and incorporating the immobilized mediators into a device whichcan be implanted subcutaneously into a patient. The reagents of theembodiments described herein may also be used with continuous analytemonitoring systems.

Alternative Embodiment A

A reagent for detecting an analyte, the reagent comprising:

a flavoprotein enzyme;

a mediator selected from the group

or a combination thereof, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹are the same or different, and are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy,aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, andcombinations thereof;

at least one surfactant;

a polymer; and

a buffer.

Alternative Embodiment B

The reagent of Alternative Embodiment A, wherein the flavoprotein enzymeis FAD-glucose dehydrogenase.

Alternative Embodiment C

The reagent of Alternative Embodiment A, wherein the mediator comprises3-(2′,5′-disulfophenylimino)-3H-phenothiazine.

Alternative Embodiment D

The reagent of Alternative Embodiment A, wherein the surfactant includesa saccharide-based surfactant or a phosphorylcholine-based surfactant.

Alternative Embodiment E

The reagent of Alternative Embodiment A, wherein the polymer is acellulose-based polymer.

Alternative Embodiment F

The reagent of Alternative Embodiment A, wherein the buffer comprises aphosphate buffer.

Alternative Embodiment G

A reagent for detecting an analyte in a fluid sample, the reagentcomprising:

FAD-glucose dehydrogenase having an activity of from about 0.1 Units/μLto about 10 Units/μL;

a 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator having aconcentration of from about 5 mM to about 120 mM;

a heptanoyl-N-methylglucamide surfactant having a concentration of fromabout 0.05 wt. % to about 0.5 wt. % of the reagent; and

a hydroxyethyl cellulose polymer having a concentration of from about0.1 wt. % to about 4 wt. % of the reagent.

Alternative Embodiment H

The reagent of Alternative Embodiment G further comprising a phosphatebuffer.

Alternative Embodiment I

The reagent of Alternative Embodiment H, wherein the phosphate bufferhas a concentration of from about 25 mM to about 200 mM and a pH of fromabout 4 to about 8.

Alternative Embodiment J

The reagent of Alternative Embodiment I, wherein the phosphate bufferhas a concentration of from about 50 mM to about 150 mM and a pH of fromabout 6 to about 7.

Alternative Embodiment K

The reagent of Alternative Embodiment G, wherein the reagent comprisesFAD-glucose dehydrogenase having an activity of from about 0.5 Units/μLto about 2.5 Units/μL, a 3-(2′,5′-disulfophenylimino)-3H-phenothiazinemediator having a concentration of from about 30 mM to about 60 mM, aheptanoyl-N-methylglucamide surfactant having a concentration of fromabout 0.1 wt. % to about 0.4 wt. % of the reagent, and a hydroxyethylcellulose polymer having a concentration of from about 0.2 wt. % toabout 0.5 wt. % of the reagent.

Alternative Embodiment L

An electrochemical test sensor comprising:

a working electrode having a surface;

a counter electrode having a surface; and

a reagent coating at least a portion of the surface of the workingelectrode and at least a portion of the surface of the counterelectrode, the reagent comprising a flavoprotein, a phenothiazine or aphenoxazine mediator, a buffer, and at least one surfactant and apolymer.

Alternative Embodiment M

The sensor of Alternative Embodiment L, wherein the flavoproteinincludes FAD-glucose dehydrogenase.

Alternative Embodiment N

The sensor of Alternative Embodiment L, wherein the phenothiazinemediator includes 3-(2′,5′-disulfophenylimino)-3H-phenothiazine.

Alternative Embodiment O

The sensor of Alternative Embodiment L, wherein the at least onesurfactant includes a heptanoyl-N-methylglucamide.

Alternative Embodiment P

The sensor of Alternative Embodiment L, wherein the polymer is acellulose-based polymer.

Alternative Process Q

A method of detecting an analyte in a fluid sample, the analyteundergoing a chemical reaction, the method comprising the acts of:

providing an electrode surface;

facilitating flow of the fluid sample to the electrode surface using atleast one surfactant;

catalyzing the chemical reaction with a flavoprotein enzyme;

generating a redox equivalent by the chemical reaction; and

transferring the redox equivalent to the electrode surface using aphenothiazine or a phenoxazine mediator.

Alternative Process R

The method of Alternative Process Q, wherein the electrode surfaceincludes a working electrode and a counter electrode, the electrodesurface including a reagent comprising the at least one surfactant, theflavoprotein enzyme, the phenothiazine mediator, and a buffer.

Alternate Process S

The method of Alternative Process R, wherein the reagent furtherincludes a cellulose-based polymer.

Alternate Process T

The method of Alternative Process S, wherein the polymer is acellulose-based polymer.

Alternate Process U

The method of Alternative Process R, wherein the buffer includes aphosphate buffer.

While the invention is susceptible to various modifications andalternative forms, specific embodiments and methods thereof have beenshown by way of example in the drawings and are described in detailherein. It should be understood, however, that it is not intended tolimit the invention to the particular forms or methods disclosed, but,to the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention.

1. A reagent for detecting an analyte, the reagent comprising: aflavoprotein enzyme; a mediator selected from the group

or a combination thereof, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹are the same or different, and are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy,aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, andcombinations thereof; at least one surfactant; a polymer; and a buffer,wherein at least one of the surfactant and the buffer includes aninorganic salt, the ratio of the total inorganic salt to mediator isless than about 3:1.
 2. The reagent of claim 1, wherein the flavoproteinenzyme is FAD-glucose dehydrogenase.
 3. The reagent of claim 1, whereinthe mediator includes 3-(2′,5′-disulfophenylimino)-3H-phenothiazine. 4.The reagent of claim 1, wherein the surfactant includes asaccharide-based surfactant or a phosphorylcholine-based surfactant. 5.The reagent of claim 1, wherein the polymer is a cellulose-basedpolymer.
 6. The reagent of claim 1, wherein the buffer includes aphosphate buffer.
 7. The reagent of claim 1, wherein the ratio of thetotal inorganic salt to mediator is in a ratio of less than about 2:1.8. A reagent for detecting an analyte in a fluid sample, the reagentcomprising: FAD-glucose dehydrogenase having an activity of from about0.1 Units/μL to about 10 Units/μL; a3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator having aconcentration of from about 5 mM to about 120 mM; a surfactant having aconcentration of from about 0.05 wt. % to about 0.5 wt. % of thereagent; a hydroxyethyl cellulose polymer having a concentration of fromabout 0.1 wt. % to about 4 wt. % of the reagent; and a buffer, whereinat least one of the surfactant and the buffer includes an inorganicsalt, the ratio of the total inorganic salt to mediator is less thanabout 2:1.
 9. The reagent of claim 8, further comprising a phosphatebuffer.
 10. The reagent of claim 8, wherein the surfactant is aheptanoyl-N-methylglucamide surfactant.
 11. The reagent of claim 8,wherein the reagent comprises FAD-glucose dehydrogenase having anactivity of from about 0.5 Units/μL to about 2.5 Units/μL, a3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator having aconcentration of from about 30 mM to about 60 mM, aheptanoyl-N-methylglucamide surfactant having a concentration of fromabout 0.1 wt. % to about 0.4 wt. % of the reagent, and a hydroxyethylcellulose polymer having a concentration of from about 0.2 wt. % toabout 0.5 wt. % of the reagent.
 12. An electrochemical test sensorcomprising: a working electrode having a surface; a counter electrodehaving a surface; and a reagent coating at least a portion of thesurface of the working electrode and at least a portion of the surfaceof the counter electrode, the reagent comprising a flavoprotein, aphenothiazine or a phenoxazine mediator, a buffer, and at least onesurfactant, a polymer and a buffer, wherein at least one of thesurfactant and the buffer includes an inorganic salt, the ratio of thetotal inorganic salt to mediator is less than about 3:1.
 13. The sensorof claim 12, wherein the flavoprotein includes FAD-glucosedehydrogenase.
 14. The sensor of claim 12, wherein the phenothiazinemediator includes 3-(2′,5′-disulfophenylimino)-3H-phenothiazine.
 15. Thesensor of claim 12, wherein the at least one surfactant includes aheptanoyl-N-methylglucamide.
 16. The sensor of claim 12, wherein theratio of the total inorganic salt to mediator is in a ratio of less thanabout 2:1.
 17. A method of detecting an analyte in a fluid sample, theanalyte undergoing a chemical reaction, the method comprising the actsof: providing an electrode surface; facilitating flow of the fluidsample to the electrode surface using at least one surfactant;catalyzing the chemical reaction with a flavoprotein enzyme; generatinga redox equivalent by the chemical reaction; and transferring the redoxequivalent to the electrode surface using a phenothiazine or aphenoxazine mediator, wherein the maximum kinetic performance is lessthan about 3 seconds.
 18. The method of claim 17, wherein the electrodesurface includes a working electrode and a counter electrode, theelectrode surface including a reagent comprising the at least onesurfactant, the flavoprotein enzyme, the phenothiazine mediator, and abuffer.
 19. The method of claim 18, wherein the reagent further includesa polymer.
 20. The method of claim 17, wherein the buffer includes aphosphate buffer.
 21. The method of claim 17, wherein the maximumkinetic performance is less than about 2 seconds.
 22. A method ofdetecting an analyte in a fluid sample, the method comprising the actsof: providing an electrode surface; providing a reagent including aflavoprotein enzyme; a mediator selected from the group

or a combination thereof wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹are the same or different, and are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy,aryloxy, nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, andcombinations thereof; at least one surfactant; and a buffer; the reagentbeing in contact with the electrode surface; contacting the fluid samplewith the reagent; and determining the concentration of the analyte,wherein the maximum kinetic performance is less than about 3 seconds.23. The method of claim 22 wherein the reagent further includes apolymer.
 24. The method of claim 22 wherein the reagent includes aphenothiazine mediator.
 25. The method of claim 22 wherein at least oneof the surfactant and the buffer includes an inorganic salt, the ratioof the total inorganic salt to mediator is less than about 3:1.
 26. Themethod of claim 25 wherein at least one of the surfactant and the bufferincludes an inorganic salt, the ratio of the total inorganic salt tomediator is less than about 2:1.
 27. The method of claim 22 wherein themaximum kinetic performance is less than about 2 seconds.